In many situations it is of vital importance to be able to detect the presence of small amounts of antibiotics. This is the case in food industries, for instance, where the increased use of antibiotics and chemotherapeutic substances in the treatment of animals has created a need for a simple, reliable and sensitive process of determination. Since antibiotics are used also in the treatment of dairy cows and since antibiotic residues in milk may both cause health hazards and be disadvantageous for food technological reasons, it is especially important to develop processes suitable for an accurate and rapid screening of milk.
Antibiotic residues in milk are generally detected by microbiological processes which utilize the fact that bacteria are able to produce acid, reduce colours and produce growth on an agar medium. These processes are based on the bactericidal, inhibitory and morphological effect of antibiotics on certain microorganisms.
The Thermocult disk technique is an agar diffusion technique which is widely used in Finland and accepted as an official antibiotic determination procedure. In this technique the test organism is B. stearothermophilus var. calidolactis. It has been developed on the basis of an IDF standard process (IDF 1970. Detection of Penicillin in Milk by a Disk Assay Technique. International Standard FIL-IDF 57. Brussels).
A process of corresponding sensitivity is disclosed by Van OS et al., Diffusion Test for the Determination of Antibiotic Residues in Milk. Neth. Milk and Dairy J. 29 (1975) 16. The Delvotest process also uses B. stearothermophilus var. calidolactis as the test organism. A sample (0.1 ml) is pipetted on agar contained in an ampoule and a tablet containing nutrients and a pH indicator is added to the ampoule. The process is based on the acid producing capability of the test organism. The ampoules are incubated at 64.degree. C. for 2 5 hours. The evaluation is based on the colour change of the agar layer.
Standard techniques further include the Intertest (BCP-Test). The test microbe used in this process is Str. thermophilus. A test tablet containing a lyophilized culture of the test microbe, nutrients, and a pH indicator (bromocresol purple) is added to a milk sample. The incubation time is 4 hours at 45.degree. C. If the sample does not contain any antibiotic, the colour of the solution turns from blue to green and further to yellow. The amount of the antibiotic can be determined to some extent on the basis of the colour by comparing to a colour map (THOROGOOD et al., An Evaluation on the Charm Test - A Rapid Method for the Detection of Penicillin in milk. J. Dairy Research 50 (1983) 185).
A drawback of these processes is their insufficient sensitivity in view of the needs of milk technology.
The determination of antibiotic residues in milk by means of chemical or physico-chemical processes is considerably less common than the use of microbiological processes. Colorimetric and chromatographic processes require skilled labour and often a complicated and expensive analyzing equipment. The processes are seldom suitable for routine analyses.
The Charm test (CHARM, S.E., A 15-minute Assay for Penicillin and other Antibiotics. Cultured Dairy Products J. 14 (1979) 24) is based on the detection of radioactivity. A lyophilized culture of B. stearothermophilus culture and lyophilized .sup.14 C-labelled penicillin are added to a sample. The amount of .sup.14 C contained in the bacterium cells is detected by a Geiger counter; the lower the penicillin concentration of the sample, the higher is the reading of the Geiger counter. The detection time is only 15 minutes and the sensitivity of the process is 0.005 I.U. of penicillin per ml. This process is also not particularly suitable for routine use since it is expensive and complicated and requires skilled persons and expensive equipment to be carried out.
Thus there is still a practical need for a sensitive process which has as broad-spectrum as possible. The process should advantageously be simple and should be capable of being carried out with equipment ready for use, whereby the test does not require specially skilled persons, but can be readily carried out, for example, on a farm.